In the manga, when the Inner Senshi and Luna went to the Moon in Act 10, they went into the ruins of the palace, and there found the Holy Blade near the Crystal Tower, inside the Chamber of Prayer. When the sword was removed from the palace floor, the image of Queen Serenity appeared before them, saying that her essence resided in the computer databanks in the ruins of the Moon Kingdom, allowing her to speak with them. She told everyone that she was the one called Selene on Earth, goddess of the Moon, or former Queen of the Moon Kingdom, Serenity.

She told them all of the past, and how the people of the Moon had long lives, given to aid their mission to protect the Silver Crystal, while at the same time watching over Earth and guiding it on a good path. Queen Serenity told them of the machinations of Queen Metalia which had led to the downfall of the Earth, and the deaths of Prince Endymion and Princess Serenity.

Serenity then told them that the dark power was reviving, because in her grief of losing her daughter, the seal she placed on Metalia was imperfect. She explained to Sailor Moon that she had to use the full power of the Silver Crystal to destroy the dark power; because the Crystal would follow her heart, she must believe in faith, unity, and love. Before she ran out of power, Queen Serenity told Sailor Moon to remember to live as a young woman because that was why she was reborn on Earth, and asked the other Senshi to protect Sailor Moon and to bring honor to the kingdom. She wished them happiness and her image faded away.

Flashbacks in later story arcs revealed more about Queen Serenity's time ruling the Silver Millenium. She granted the respective planetary castles to Sailor Neptune, Sailor Uranus, and Sailor Pluto upon their birth. She likewise instructed Pluto on the responsibilities of her role as the guardian of space-time, informing her of the three taboos of her station: traveling through time, abandoning her post at the Space-Time Door, and stopping time.

When they fought Queen Nehellenia, the Sailor Senshi learned that Queen Serenity first encountered her during the celebration of Princess Serenity's birth. Nehellenia had infiltrated the Moon, living deep within it, and though Serenity welcomed her so long as she sought peace, it was not to be. Nehellenia asked Serenity to join her instead, mentioning that they had both originated from a distant place, possibly meaning the Galaxy Cauldron. Queen Serenity used the power of the Silver Crystal to seal Nehellenia into her ornate mirror, within which the Queen of the Dead Moon dwelt until encountering the Sailor Senshi.

After Eternal Sailor Moon saved the galaxy by resetting the Galaxy Cauldron and dispersing Chaos, Guardian Cosmos revealed that when Queen Serenity died, she returned to the Galaxy Cauldron.

Queen Serenity's only appearance in the live-action series was in the Special Act. She first appeared when Luna called her to express concern that Usagi and Mamoru's relationship seemed to be falling apart. Queen Serenity assured Luna that things would be sure to work themselves out in due course, but warned her of a dark presence that was trying to awaken. She appeared again later, when Luna and Makoto were concerned about how to deal with the growing threat of Queen Mio. Queen Serenity sent them the Legendary Sword, which she called the Spirit of the Senshi, in order to revive the Senshi's powers.

Like the live-action version of Princess Serenity, Queen Serenity did not wear the odango hairstyle. Her dress was different than that of her anime and manga counterparts, and unlike them, she also wore a crown. Queen Serenity only refered to Princess Serenity as "the Princess" and never as "Serenity."

Serenity's name is derived from the Greek Moon goddess Selene, who fell in love with the mortal man Endymion. In Sailor Moon, the original Greek myth is divided between Queen Serenity (the goddess) and Princess Serenity (who fell in love with Endymion).

Although Queen Serenity was blonde in her first anime appearance in episode 35, her hair was silver all of the other times she appeared.

In the opening sequence of the first five episodes of Sailor Moon Crystal, Queen Serenity had blonde hair and dark blue eyes, but as of the opening for Act 6 she appeared with white hair and grey eyes.